The phosphorylated derivatives of phosphatidylinositol (PtdIns), collectively called phosphoinositides (PIs), are used by eukaryotic cells as distinct second messengers that regulate a myriad of cellular responses. The ability of PIs to control such diverse functions is based on the spatially- and temporally- controlled enzymatic activities that add one, two or three phosphate groups at different sites of the PtdIns head group. PtdIns 5-P and PtdIns 3,5-P2 represent a recent key addition to the group of PIs in mammalian cells. The mechanism of their biosynthesis, basal levels, regulation, specific function and effector targets are largely unknown. In yeast, PtdIns 3,5-P2, but not PtdIns 5-P, has been found and described to be important in cell growth and selective cargo sorting into the vacuole lumen. The long-term goal of this project is to determine how the mammalian biosynthesis of PtdIns 5-P and PtdIns 3,5-P2 can be modulated for preventive and therapeutic purposes. To this end, we have identified and characterized a novel mammalian protein, PIKfyve, which is an active PtdIns 5-P- and PtdIns 3,5-P2- producing kinase in vitro. The objective of this application is to determine to what degree the PIKfyve enzymatic activity contributes to PtdIns 5-P and PtdIns 3,5-P2 biosynthesis in mammalian cells and to identify factors that spatially and temporally regulate their intracellular production and function. The central hypothesis of the application is that PIKfyve is an active 5'-phosphoinositide kinase in the cellular context, whose products are essential in maintaining endomembrane homeostasis and are involved in the signaling pathways of hormones and growth factors. Our hypothesis is based on strong preliminary data, including a characterization of dramatic phenotypic changes induced upon expression of a dominant-negative kinase-dead PIKfyve point mutant in different cells and recruitment of cytosolic PIKfyve populations to inner membranes upon acute cell stimulation with insulin. Our specific aims address four critically important issues: 1) the identity and downstream protein targets of the PIKfyve lipid products in intact cells; 2) the general role of PIKfyve enzymatic activity in maintaining cell morphology and endomembrane homeostasis; 3) the function of PIKfyve in the intracellular signal transduction mechanisms exemplified by insulin-induced transmission; 4) the identity of the molecular elements and mechanisms that regulate, or are regulated by PIKfyve intracellular function. These key questions will be addressed by exploiting the most appropriate and sensitive techniques including adenovirus- mediated gene delivery, microinjection into intact cells, and optimized HPLC separation of cellular PIs. The proposed work is innovative because it focuses on a novel PI enzymatic activity, recently identified by our group and will provide the first evidence for PtdIns 5-P and PtdIns 3,5-P2 biosynthesis, regulation and role in mammalian cells. Our studies are expected to fundamentally advance the field of phospholipid intracellular signaling and to eventually provide new targets for preventive and therapeutic intervention, particularly to cancer and diabetic patients.